The present invention relates to a new protein, protein SIC, that can be derived from Streptococcus pyogenes strains of serotypes M1 and M57. Methods for analysis and purification and pharmaceutical preparations including vaccine compositions related to the protein as well as specific antibodies are also claimed.
Streptococcus pyogenes is an important human pathogen causing a number of acute suppurative infections such as erysipelas, necrotizing fasciitis, and pharyngitis. These Gram-positive bacteria also cause a serious toxic shock-like syndrome, whereas glomreulonephritis and rheumatic fever are serious poststreptococcal sequelae.
To elude the host defence and establish an infection, S. pyogenes has developed multiple molecular mechanisms. Some of these are dependent on genes located in a chromosomal region designated the mga locus according to a recent agreement, which is under the control of the positive regulator gene mga, previously called mry (Caparon and Scott, 1987: Proc. Natl. Acad. Sci. U. S. A. 84, 8677-8681) or virR (Simpson et al., 1990: J. Bacteriol. 172, 696-700).
Since the late 1980""s unusually severe S. pyogenes infections have been reported world-wide. These hyperacute and often lethal infections have frequently been associated with the M1 serotype (Musser et al., 1993: J. Infect. Dis. 167, 337-346). This serotype is also connected with glomerulonephritis and rheumatic fever.
Based on structural variations in the antiphagocytic M protein (Fischetti, 1989: Clin. Microbiol. Rev. 2, 285-314; and Kehoe, 1994: New Compr. Biochem. 27, 217-261) S. pyogenes can be divided into more than 80 different serotypes. Most of these serotypes are rather harmless. However, during the last years, a lot of lethal streptococcal infections have been caused by strain belonging to the M1 serotype. Presently, because of the large amount of serotypes and the great similarity between these types, it is time-consuming and labourious to determine the serotype of a sample of Streptococcus pyogenes. Consequently, there is a need for simpler methods for determining whether a particular sample of Streptococcus pyogenes is virulent. There is also a need for better vaccines against virulent Streptococcus pyogenes strains.
A new protein from Streptococcus pyogenes, serotype M1, fulfilling the above mentioned needs has now been discovered and characterized. The protein and its corresponding gene has been obtained from strain AP1, which is of the M1 serotype.
In the mga locus of AP1, the regulatory gene mga is followed by emm1, the gene encoding the M1 protein. Immediately down-stream of emm1 is sph ({dot over (A)}kesson et al., 1994: Biochem. J. 300, 877-886), the gene encoding an IgGFc-binding M protein-related molecule called protein H ({dot over (A)}kesson et al., 1990: Mol. Immunol. 27, 523-531; Gomi et al., 1990: J. Immunol. 144, 4046-4052; WO 91/19740; EP 0 371 199). Located adjacent to sph is a previously uncharacterized gene. This gene constitutes one of the objects of the present invention.
The protein shows some variability and M1 serotype strains of Streptococcus pyogenes producing variants of protein SIC have also been isolated. When comparing the amino acid sequences of three different protein SIC variants, some conserved regions were found.
The protein encoded by the uncharacterized gene as well as variants, subfragments and multiples of the protein having essentially the same antigenic and/or binding characteristics also constitutes an object of the present invention. The data obtained implicate that this extracellular protein plays a role in S. pyogenes pathogenicity and virulence through previously unknown molecular mechanisms. The new protein is referred to as protein SIC, Streptococcal Inhibitor of Complement-mediated lysis.
xe2x80x9cSIC proteinsxe2x80x9d as utilized herein refers to streptococcal proteins which inhibit hemolysis by interacting with the plasma proteins clusterin and members of the cystatin protein superfamily, such as HRG, as demonstrated by affinity chromatography on a protein SIC Sepharose column or by indirect ELISA. SIC proteins may be distinguished from other proteins based upon criteria such as specific binding to the above mentioned plasma proteins and sequence homology. For example, SIC proteins of the present invention should comprise at least one of the following partial amino acid sequences:
a) glu thr tyr thr ser arg asn phe (SEQ ID NO:7);
b) asp trp ser gly asp asp trp pro glu asp asp trp (SEQ ID NO:8);
c) arg ser gly val gly leu ser gln tyr gly trp ser (SEQ ID NO:9);
d) trp ser ser asp lys lys asp glu thr glu asp lys thr (SEQ ID NO:10);
e) gly thr gly tyr glu lys arg asp asp trp gly gly pro gly (SEQ ID NO:11);
f) lys arg asp asp trp arg gly pro gly his ile pro lys pro (SEQ ID NO:12);
preferably the amino acid sequence of the SIC proteins should be at least 70% homologous, more preferably at least 85% homologous, still more preferably at least 90% homolgous and most preferably at least 95% homologous to anyone of the amino acid sequences disclosed in SEQ. ID. NOS 2, 3 and 4.
As already mentioned and suggested above, different variants of protein SIC have been isolated. Most isolates produced a variant showing close resemblance to protein SIC isolated from strain AP1, whose amino acid sequence is disclosed in SEQ. ID. NO. 2. However, two more variants having the amino acid sequences according to SEQ. ID. NO. 3 and SEQ. ID. NO. 4, respectively, have also been discovered. After aligning the sequences of the different variants the conserved partial sequences a)-f) above were found. Regions a)-e) are present in all known protein SIC variants. Protein SIC from one of the isolates only comprised a part of the f) region. Consequently the above regions are considered to have important functions in the protein.
By xe2x80x9csubfragmentxe2x80x9d is meant a part-fragment of the given protein having essentially the same antigenic and/or binding characteristics. By xe2x80x9cvariantsxe2x80x9d is meant proteins or peptides in which the original amino acid sequence has been modified or changed by insertion, addition, substitution, inversion or exclusion of one or more amino acids. By xe2x80x9cmultiplesxe2x80x9d is meant those proteins containing multiples of the whole original protein or those protein containing multiples of subfragments and/or variants thereof.
The present invention also relates to nucleic acid sequences encoding protein SIC. As utilized within the context of the present invention, nucleic acid sequences which encode protein SIC are deemed to be substantially similar to those disclosed herein if: (a) the nuclcic acid sequence is derived from the coding region of a native protein SIC gene (including, for example, variations of the sequences disclosed herein); (b) the nucleic acid sequcnce is capable of hybridization to nucleic acid sequences of the present invention under conditions of either moderate or high stringency (hybridization in 5xc3x97SSPE containing 0.1% SDS and 0.1 mg/ml ssDNA, at 50-65xc2x0 C. dependent on the probe length, or 10-20xc2x0 C. below the Tm of the probe; washing in 1xc3x97SSPE, 0.1% SDS at 15-20xc2x0 C. below the Tm of the probe for moderate stringency, and in 0.1xc3x97SSPE, 0.1% at 10xc2x0 C. below the Tm of the probe for high stringency conditions) (see Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, NY, 1989); or (c) nucleic acid sequences are degenerate as a result of the genetic code to the nucleic acid sequences defined in (a) or (b). Furthermore, although nucleic acid molecules are primarily referred to herein, as should be evident to one of skill in the art given the disclosure provided herein, a wide variety of related nucleic acid molecules may also be utilized in various embodiments described herein, including for example, RNA, nucleic acid analogues, as well as chimeric nucleic acid molecules which may be composed of more than one type of nucleic acid.
Within another aspect of the present invention, probes and primers are provided for detecting nucleic acids scquences which encode protein SIC. Within one embodiment of the invention, probes are provided which are capable of hybridizing to protein SIC nucleic acids (DNA or RNA}. For purposes of the present invention, probes are xe2x80x9ccapable of hybridizingxe2x80x9d to protein SIC nucleic acids if they hybridize to Sequence I.D. No 1, 5 or 6 under conditions of moderate or high stringency (see the section above concerning nucleic acid molecules, and Sambrook et al., supra); Preferably, the probe may be utilized to hybridize to suitable nucleotide sequences in the presence of 5xc3x97SSPE, 0.1% SDS, and 0.1 mg/ml ssDNA at 10-20xc2x0 C. below the Tm of the probe. Subsequent washes may be performed in 1xc3x97SSPE, 0.1% SDS at 15-20xc2x0 C. for conditions of moderate stringency, and in 0.1xc3x97SSPE, 0.1% SDS at 10xc2x0 C. below the Tm of the probe for conditions of high stringency.
Probes of the present invention may be composed of either deoxyribonucleic acids (DNA) ribonucleic acids (RNA), nucleic acid analogues, or any combination of these, and may be as few as about 12 nucleotides in length, usually about 14 to 18 nucleotides in length, and possibly as large as the entire sequence which encodes protcin SIC. Selection of probe size is somewhat dependent upon the use of the probe. For example, a long probe used under high stringency conditions is more specific, whereas a oligonucleotide carefully selected from the sequence can detect a structure of special interest.
Probes may be constructed and labeled using techniques which are well known in the art. Shorter probes of, for example, 12 or 14 bases may be generated synthetically. Longer probes of about 75 bascs to less than 1,5 kb are preferably generated by, for example, PCR amplification in the presence of labeled precursors such as 32P-dCTP, digoxigenin-dUTP, or biotin-dATP. Probes of more than 1.5 kb are generally most easily amplified by transfecting a cell with a plasmid containing the relevant probe, growing the transfected cell into large quantities, and purifying the relevant sequence from the transfected cells (see Sambrook et al., supra).
Probes may be labeled by a variety of markers, including, for example, radioactive markers, fluorescent markers, enzymatic markers, and chromogenic markers. The use of 32P is particularly preferred for marking or labeling a particular probe.
As noted above, nucleic acid probes of the present invcntion may be utilized to detect the presence of protcin SiC nucleic acid molecules within a sample. However, if such nucleic acids molecules are present in only a limited number, then it may be beneficial to amplify the relevant sequence such that it may be more readily detected or obtained.
A variety of methods may be utilized in order to amplify a selected sequence, including, for example, RNA amplification (see Lizardi et al., Bio/Technology 6:1197-1202, 1988; Kramer et al., Nature 339:401-402, 1989; Lomeli et al., Clinical Chem. 35(91) 1826-1831, 1989; U.S. Pat. No. 4,786,600), and nucleic acid amplification utilizing Polymerase Chain Reaction (xe2x80x9cPCRxe2x80x9d) (see U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159) (see also U.S. Pat. Nos. 4,876,187, and 5,011,769, which describe an alternative detection/amplification system comprising the use of scissile linkages). Within a particularly preferred embodiment, PCR amplilication is utilized to detect or obtain protein SIC nucleic acids. Briefly, as described in greater detail below, a nucleic acid sample is denatured at 95xc2x0 C. in order to generate single stranded nucleic acid. Specific primers, as discussed below, are then annealed at 370xc2x0 C. to 70xc2x0 C., depending on the proportion of AT/GC in the primers. The primers are extended at 72xc2x0 C. with Taq polymerase in order to generate the opposite strand to the template. These steps constitute one cycle, which may be repeated in order to amplify the selected sequence.
Primers for the amplification of a selected sequence should be selected from sequences which are highly specific and form stable duplexes with the target sequence. The primers should also be non-complementary, especially at the 3xe2x80x2 end, should not form dimers with themselves or other primers, and should not form secondary structures or duplexes with other regions of nucleic acid. In general, primers of about 18 to 20 nucleotides are preferred, and may be easily synthesized using techniques well known in the art.
In another aspect, the present invention relates to vectors and host cells comprising the above mentioned nucleic acid sequences. The above described nucleic acid molecules which encode protein SIC (or portions thereof) may be readily introduced into a wide variety of host cells. Representative examples of such host cells include plant cells, eukaryotic cells, and prokaryotic cells. Within preferred embodiments, the nucleic acid molecules are introduced into cells from a vertebrate or warm-blooded animal, such as a human, macaque, dog, cow, horse, pig, sheep, rat, hamster, mouse, or a fish, or any hybrid thereof.
The nucleic acid molecules (or vectors) may be introduced into host cells by a wide variety of mechanisms, including for example calcium phosphate-mediated transfection (Wigler et al., Cell 14:725, 1978), lipofection; gene gun (Corsaro and Pearson, Somatic Cell Gen. 1:603, 1981; Graham and Van der Eb, Virology 52:456, 1973), electroporation (Neumann et al,. EMBO J. 1:841-845, 1982), retroviral, adenoviral, protoplast fusion-mediated transfection or DEAE-dextran mediated transfection (Ausubel et al., (eds.), Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, N.Y., 1987).
The nucleic acid molecules, antibodies, and proteins of the present invention may be labeled or conjugatad (either through covalent or non-covalent means) to a variety of labels or other molecules, including for example, fluorescent markers, enzyme markers, toxic molecules, molecules which are nontoxic but which become toxic upon exposure to a second compound, and radionuclides.
Representative examples of fluorescent labels suitable for use within the present invcetion include, for example, Fluorescein Isothiocyanate (FITC), Rodamine, Texas Red, Luciferase and Phycoerythrin (PE). Particularly preferred for use in flow cytometry is FITC which may be conjugated to purified antibody according to the method of Keltkamp in xe2x80x9cConjugation of Fluorescein Isothiocyanate to Antibodies. I. Experiments on the Conditions of Conjugation,xe2x80x9d Immunology 18:865-873, 1970. (See also Keltkamp, xe2x80x9cConjugation of Fluoresscein Isothiocyanate to Antibodies. II. A Reproducible Method,xe2x80x9d Immunology 18:875-881, 1970; and Goding, xe2x80x9cConjugation of Antibodies with Fluorochromes: Modification to the Standard Methods,xe2x80x9d J. Immunol. Methods 13:215-226, 1970). For histochemical staining, HRP, which is preferred, may be conjugated to the purified antibody according to the method of Nakane and Kawaoi {xe2x80x9cPeroxidase-Labeled Antibody: A New Method of Conjugation,xe2x80x9d J. Histochem. Cytochem. 22:1084-1091, 1974; see also, Tijssen and Kurstak, xe2x80x9cHighly Efficient and Simple Methods for Preparation of Peroxidase and Active Peroxidase Antibody Conjugates for Enzyme Immunoassays,xe2x80x9d Anal. Biochem. 136;451-457, 1984).
Representative examples of enzyme markers or labels include alkaline phosphatase, horse radish peroxidase, and xcex2-galactosidase. Representative examples of toxic molecules include ricin, abrin, diphtheria toxin, cholera toxin, gelonin, pokeweed antiviral proteein, tritin, Shigella toxin, and Pseudomonas exotoxin A. Representative examples of molecules which are nontoxic, but which become toxic upon exposure to a second compound include thymidine kinases such as HSVTK and VZVTK. Representative examples of radionuclides include Cu-64, Ga-67, CTa-68, Zr-89, Ru-97, Tc-99m, Rh-105, Pd-109, ln-111, I-123, I-125, I-I31, Re-186, Re-188, Au-I98, Au-199, Pb-203, At-211, Pb-212 and Bi-212.
As will be evident to one of skill in the art given the disclosure provided herein, the above described nucleic acid molecules, antibodies, proteins and peptides may also be labeled with other molecules such as colloidal gold, as well either member of a high affinity binding pair (e.g., avidin-biotin).
As noted above, the present invention also provides a variety of pharmaceutical compositions, such as vaccine compositions against certain streptococcal infections, comprising one of the above described anti-protein SIC antobodies, or protein SIC (or a peptide portion thereof), along with a pharmaceutically or physiologically acceptable carrier, excipients or diluents. Generally, such carriers should be nontoxir, to recipients at the dosages and concentrations employed. Ordinarily, the preparation of such compositions entails combining the therapeutic agent with buffers, antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, amino acids, carbohydrates including glucose, sucrose or dextrins, chelating agents such as EDTA, glutathione and other stabilizers and excipients. Neutral buffered saline or saline mixed with nonspecific serum albumin are exemplary appropriate diluents.
In addition, the pharmaceutical compositions of the present invention may be prepared for administration by a variety of different routes, including for example intraarticularly, intracranially, intradermally, intramuscularly, intraocularly, intraperitoneally, intrathecally, intravenously, subcutaneously or even directly into a tumor (for example, by stereotaxic injection). In addition, pharmaceutical compositions of the present invention may be placed within containers, along with packaging material which provides instructions regarding the use of such pharmaceutical compositions. Generally, such instructions will include a tangible expression describing the reagent concentration, as well as within certain embodiments, relative amounts of excipient ingredients or diluents {e,g., water, saline or PBS) which may be necessary to reconstitute the pharmaceutical composition.
As previously mentioned, protein SIC is involved in S. pyogenes pathogenicity and moreover, it is an extracellular protein. Hence, by carrying out an analysis of protein SIC, it is possible determine the presence of virulent Streptococcus pyogenes bacteria in a sample. Consequently, other objects of the present invention relates to methods and kits for analysis of protein SIC and/or corresponding antibodies based on for example enzyme-linked immonosorbent assy (ELISA), radioimmunological assay (RIA) and the polymerase chain reaction (PCR).
As previously mentioned, the present patent application relates to protein SIC, a novel extracellular protein of group A streptococci. Analogous to other proteins encoded by genes under control of mga, protein SIC contains repeated sequences. However, the sequence of protein SIC, including the repeats, shows no homology to previously sequenced genes. It is also noteworthy that in contrast to all other described products of the mga regulon, protein SIC does not have the typical structural features of cell wall proteins in Gram-positive bacteria; i.e. a COOH-terminal region anchored to the cell wall through an LPXTG motif (Fischetti et al., 1990: Mol. Microbiol. 4, 1603-1605; Schneewind et al., 1995: Science 268, 103-106; Schneewind et al., 1992: Cell 70, 267-281), followed further towards the COOH-terminus by a hydrophobic membrane-spanning domain and a tail of mostly positively charged amino acid residues. The missing cell wall anchor, the occurrence of a typical signal sequence, and the fact that considerable amounts of protein SIC is found in the growth medium, suggest that the molecule is secreted and has extracellular function(s).
Previous work has demonstrated several interactions between components of the complement system and proteins encoded by genes of the mga regulon. Members of the M protein family (M protein, protein Arp, protein Sir, and protein H) have been reported to bind complement factor H, CD46, and/or the C4b-binding protein (Horstmann et al., 1988: Proc. Natl. Acad. Sci. U. S. A. 85, 1657-61; Okada et al., 1995: Proc. Natl. Acad. Sci. U. S. A. 92, 2489-2493; Thern et al., 1995: J. Immunol. 154, 375-386; WO 91/19740; EP 0 371 199; EP 0 367 890), three proteins with regulatory functions in the complement system. Furthermore, the C5a peptidase (Wexler et al., 1985: Infect. Immun. 39, 239-246) which can be released from the streptococcal cell wall by a cysteine proteinase produced by the bacteria (Berge et al., 1995: J. Biol. Chem. 270, 9862-9867), cleaves the C5-derived fragment C5a and destroys its chemoattractant activity for polymorphonuclear leukocytes (Wexler et al., 1985: Proc. Natl. Acad. Sci. U. S. A. 82, 8144-8148).
The specific interactions between protein SIC and the plasma proteins clusterin and HRG resulted in a study of the final cytolytic step in the complement cascade. Clusterin is known to inhibit the hemolytic activity of complement by binding to MAC (Membrane Attack Complex) (Tschopp et al., 1993: J. Immunol. 151, 2159-2165; Tschopp et al., 1994: Clin. Exp. Immunol. 97 suppl 2, 11-14), whereas the influence of HRG on MAC is biphasic, inhibitory or stimulatory, depending on the experimental conditions (Chang et al., 1992: Blood 79, 2973-2980). As demonstrated here, protein SIC was inhibitory to hemolysis in classical pathway as well as alternative pathway systems. Furthermore, protein SIC was shown to be incorporated into C5b-C9 complexes formed in serum. Although other mechanisms may also be considered, the findings suggest that the anticomplementary action af protein SIC is focused on the terminal cytotoxic functions of complement.
The metabolically inert erythrocyte is a very sensitive target for MAC whereas most pathogenic bacteria including streptococci are resistant to complement-mediated cytolysis. However, apart from its cytolytic activity, MAC also has pro-inflammatory effects by stimulating the production and release of inflammatory mediators such as reactive oxygen metabolites, metabolites of arachidonic acid, and cytokines (Morgan 1989: Biochem. J.: 264, 1-14). Bacterial products affecting the various functions of MAC, directly or indirectly, could therefore influence the host-parasite relationship. The molecular complexity necessary to establish and maintain this relationship, makes it difficult to predict the consequences of any isolated interaction. However, in the case of pathogenic and virulent bacteria like S. pyogenes, the balance is disturbed, and there is circumstantial evidence that protein SIC may contribute to the imbalance of the host-parasite relationship in S. pyogenes infections.
To our knowledge protein SIC is the first bacterial protein reported to interact with clusterin or HRG. As mentioned, glomerulonephritis represents a medically significant sequelae following infections with S. pyogenes. In these cases certain M serotypes are more common, including the two protein SIC-producing serotypes M1 and M57 (Holm, 1988: Acta Pathol. Microbiol. Scand. 96, 189-193). In post-streptococcal glomerulonephritis immunoglobulin deposits are found in the glomeruli. Interestingly, MAC is regarded as a mediator of glomerular injury in immune complex-related disease (Couser et al., 1985: Kidney Int. 28, 879-890), and clusterin was found to be co-localized with MAC in biopsies from glomerulonephritic kidneys (French et al., 1992: Clin. Exp. Immunol. 88, 389-393).
Also, plasma depleted of clusterin (to  less than 30%) enhanced proteinuria and deposition of MAC components in perfused kidneys (Saunders et al., 1994: Kidney Int. 45, 817-827). The association of protein SIC to nephritogenic M serotypes, and its binding of clusterin in human plasma, makes it interesting to test if protein SIC can induce kidney damage in an animal model.
Since the late 1980""s a world-wide increase of hyperacute, toxic, and often lethal S. pyogenes infections, has attracted also public attention (Nowak, 1994: Science 264, 1665). These systemic infections have particularly been associated with streptococci of the M1 serotype, and the observation that all M1 strains tested, including isolates from Swedish patients with toxic and severe infections (Holm et al., 1992: J. Infect. Dis. 166, 31-37), carry and express the sic gene, supports the notion that protein SIC plays a role in pathogenicity and virulence. The present invention may therefor be used for preventing and/or treating S. pyogenes infections.
As previosly mentioned, effective amounts of the protein or fragments or variants thereof can be used as active ingredients in pharmaceutical compositions, especially in vaccine compositions against streptococcal infections, possibly together with pharmaceutically acceptable adjuvants and excipients. Suitable pharmaceutically acceptable adjuvants are those conventionally used in this field. Examples of suitable excipients are mannitol, lactose, starch, cellulose, glucose, etc., only to mention a few. The examples given of the adjuvant and the excipients are not to be regarded as limiting the invention.
The invention will now be described in more detail with reference to the accompaying drawings, in which